Project Summary Biological insecticides are used worldwide for control of human disease vectors. Among these is Bacillus thu- ringiensis israelensis (Bti), which has been used for many decades for mosquito and blackfly control with no field resistance, primarily due to the presence of three Cry and two Cyt toxins. Our long-term goal is to under- stand the precise mechanisms of action of these Cry and Cyt toxins, and how the mosquito responds to this bacterial agent. Such knowledge will aid in the lasting use of Bti for control of important insect vectors of hu- man diseases. But there are significant knowledge gaps in our understanding of Bti action. We plan to address three of these knowledge gaps in this proposal. First, our preliminary data shows that the Bti Cry11A toxin binds with high affinity to a novel target, an N- cadherin, which is down regulated in a Cry11A-resistant strain. We will test the hypothesis that Cry11Aa dis- rupts midgut intercellular junctions by binding and then disrupting intercellular junctions. Hence N-Cad protein domains that bind Cry11Aa toxin will be identified, and evaluated if they can also bind the Cry4Aa and Cry4Ba toxins. We will evaluate the effect of N-Cad in vivo silencing on Cry11Aa toxicity will be evaluated and finally the expression of this protein in midgut junctions analyzed. This novel mechanism will show Bti toxins can dis- rupt midgut function through a previously unidentified mechanism, i.e. paracellular transport. Confirmation of this mechanism would suggest that Bti action is even more complex than currently understood, and like other pathogenic bacteria and their toxins utilize disruption of these junctions to circumvent the midgut epithelial bar- rier to exert their toxicity. Second, the precise mechanism by which the Cyt toxin inserts into membranes and synergizes toxicity is not known. We will test the hypothesis that the Cyt1A alpha helices 1 and 3, are critical for toxin oligomerization and membrane insertion, and while other domains are essential for synergism. We will determine the ability of these helix mutants to oligomerize and synergize toxicity, and then analyze the Cry11Aa structure that inserts into the membrane. Third, we show that the mosquito mounts a cellular defense response against bacterial toxins. But the mechanism by which this occurs is not known. Our recent RNA seq data from larvae exposed to Cry11A toxin and of a Cry11A-resistant line show that of many genes altered, a number are in the MAPK signaling pathway. Thus we will test the hypothesis that induction of this pathway en- ables larvae to mount a defense thereby being less susceptible to Bti. Significantly this project continues a highly successful long-term collaboration between three different labora- tories (Gill, Bravo and Soberon) to best use available resources and expertise of each laboratory. We have re- cruited a bioinformaticist (Girke) to aid our analyses.